Cassette for facilitating optical sectioning of a retained tissue specimen

ABSTRACT

A cassette for retaining a specimen of surgically exposed tissue from a patient in an orientation that facilitates optical sectioning of the tissue by a confocal microscopic or other optical imaging microscope. The cassette includes a base member having a rigid optically transparent window upon which a tissue specimen is situated, a pliable membrane locatable over a substantial portion of the base member including the window, and an upper member, having an aperture therethrough, which can cover the base member to provide an enclosed cavity between the membrane and the window sealing the tissue specimen therein. The edges of the tissue specimen may be positioned planar against the window and retained in that position by bonds formed between the membrane and window at multiple points or locations around the tissue specimen. The specimen retained in the cavity is imagible by a microscope through the window of the base member.

This application is a divisional of U.S. patent application Ser. No.10/162,317, filed Jun. 4, 2002, now U.S. Pat. No. 8,149,506, which is acontinuation of U.S. patent application Ser. No. 09/502,252, filed Feb.17, 2000, now U.S. Pat. No. 6,411,434, which claims the benefit ofpriority to U.S. Provisional Application No. 60/120,534, filed Feb. 17,1999, which is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a cassette for retaining a tissuespecimen, and relates particularly to a cassette for retaining aspecimen of surgically exposed tissue from a patient in an orientationthat facilitates optical sectioning of the tissue by a confocalmicroscope, or other optical imaging microscope, for Mohs micrographicsurgery. The invention further relates to a method of using the cassettefor preparing a tissue specimen for examination by a confocal microscopeor other optical imaging microscope, and a system for opticallysectioning a tissue specimen retained in a cassette.

BACKGROUND OF THE INVENTION

In Mohs micrographic surgery, tissue having a tumor, typically acarcinoma on the skin of the head or neck, is excised from a patientunder microscopic control. The excised tissue specimen, often called abiopsy, is horizontally sliced to provide tissue sections which are thenhistologically prepared on slides. The slides are reviewed under amicroscope to determine whether the tumor is fully contained in theexcised tissue. This is indicated by the absence of the tumor in theedges or margins of the excised tissue. If the tumor is not fullycontained in the excised tissue, additional tissue from the patient isexcised and the procedure repeated until all tissue sections takenindicate the tumor has been removed from the patient. Mohs surgerypermits removal of a tumor with maximum preservation of normalsurrounding tissue. Mohs surgery is described in the book entitled MOHSSURGERY FUNDAMENTALS AND TECHNIQUES (Kenneth G. Gross, M. D. et al.eds., 1999).

To prepare each tissue specimen in Mohs surgery, multiple sections orslices are manually made with a microtome, where each section is planarand parallel to each other. Often the tissue specimen is first frozen tomake the tissue easier to manipulate and cut by the microtome. However,since numerous sections must be made from each tissue specimen and thenhistologically prepared on slides, this procedure is both tedious andtime consuming.

U.S. Pat. No. 4,752,347 provides a method and apparatus for preparing atissue specimen for sectioning for Mohs surgery. The patent describesplacing an excised tissue specimen on a platform, applying a flexibleplastic membrane over the tissue specimen, and evacuating the areabetween the membrane and the tissue specimen. This retracts the membraneonto the platform and pushes the edges of the tissue specimen into aplanar orientation parallel to the platform. While under the pressure ofthe membrane, the tissue sections may be manipulated by an operatorthrough the membrane until the desired orientation is obtained. Theedges of the tissue specimen are thus oriented to flatten the edges ofthe specimen down. The specimen is then frozen, peeled away from theplatform, and sectioned by a microtome. Since the edges of the specimenare oriented planar when sectioned by the microtome, a single sectioncan be made having the edges of interest in Mohs surgery. This procedureis adequate for obtaining a section which can be placed on a slide forreview under a microscope, but is not useful with optical imagingtechniques, such as provided by confocal microscopes, which can examinea surgically exposed tissue specimen without the need for traditionalmicrotome sectioning or slide preparation.

Confocal microscopes optically section naturally or surgically exposedtissue to produce microscopic images of tissue sections. An example of aconfocal microscope is the “VivaScope” manufactured by Lucid Inc. ofHenrietta, N.Y.. Other examples of confocal microscopes are described inU.S. Pat. No. 5,788,639, published International Patent Application WO96/21938, and in articles by Milind Rajadhyaksha et al., “In vivoConfocal Scanning Laser Microscopy of Human Skin: Melanin providesstrong contrast,” The Journal of Investigative Dermatology, Volume 104,No. 6, June 1995, and Milind Rajadhyaksha and James M. Zavislan,“Confocal laser microscope images tissue in vivo,” Laser Focus World,February 1997, pages 119-127. Further, optically sectioned microscopicimages of tissue can be produced by optical coherence tomography orinterferometry, such as described in Schmitt et al., “Opticalcharacterization of disease tissues using low-coherence interferometry,”Proc. of SPIE, Volume 1889 (1993), or by a two-photon laser microscope,such as described in U.S. Pat. No. 5,034,613.

One problem with optical imaging of a tissue specimen for Mohs surgeryis that the tissue specimen is generally too thick, for example 2-3 mm,to enable optically imaging of the edges of the specimen to determine ifthe specimen contains all of the tumor. Typically, a confocal microscopeis limited to producing adequate images of tissue sections at 100-200microns. Thus, it would be desirable to optically image a tissuespecimen in which the edges of the tissue specimen are oriented planaragainst an optically transparent surface through which the specimen canbe optically sectioned.

In addition, optical imaging systems, such as confocal microscopes,generally require the use of a liquid immersion objective lens directedtoward the tissue specimen. This necessitates that the tissue specimenbe wetted or immersed in a fluid having an optical index suitable forthe objective lens, otherwise, the imaging performance of the system isseverely degraded. It is thus also desirable that fluids may beinsertable to a properly oriented tissue specimen, and furtherremovable, such that the fluid may be replaced with another fluid tochange the imaging characteristics of the tissue.

Although U.S. Pat. No. 4,752,347 describes positioning the edges of atissue specimen planar on a platform under a plastic membrane held byvacuum to the platform, the tissue specimen described in this patent ismechanically sectioned, rather than optically sectioned. Further, priorto mechanical sectioning, such a specimen is incompatible with opticalimaging techniques since fluid cannot be present with a tissue specimenin a vacuum, and the platform does not provide a surface through whichoptical imaging can be performed. For example, liquids under a vacuumwould be suctioned away from the specimen, while gases, under thereduced pressure, would dissolve in any liquids to form bubbles, or suchgases may boil or evaporate.

Furthermore, the traditional slides from tissue specimens produced byMohs surgery must be archived for a minimum retention time in compliancewith regulatory requirements, or to enable future reanalysis of theslides for legal purposes. This requires storage of the slides for manyyears which is cumbersome for large volumes of sectioned tissuespecimens. Furthermore, each slide must be labeled in accordance with anidentification system to facilitate locating the slides if they are everneeded.

In the area of dialysis, U.S. Pat. No. 5,503,741 describes a devicehaving a sealed vacant chamber formed between two parallel dialysismembranes affixed to each side of a gasket. A needle may be insertedthrough the gasket to delivery or withdraw a sample from the chamber.The device is used for permitting dialysis, i.e., molecular exchange,between the sample in the chamber and an external solution. Such adevice is limited to dialysis and provides no mechanism for retaining atissue specimen in a planar orientation for optical sectioning.

SUMMARY OF THE INVENTION

Accordingly, it is the principal object of the present invention toprovide a cassette for retaining a specimen of surgically exposed tissuefrom a patient which facilitates optical examination of the tissue by aconfocal microscopic or other optical imaging microscope.

It is another object of the present invention to provide a cassette forretaining a tissue specimen in a cavity in which the edges of the tissuespecimen can be positioned and retained in a planar orientation againstan optically transparent surface

It is still another object of the present invention to provide acassette for retaining a tissue specimen in a cavity which allows fluidto be insertable and removable from the cavity containing the tissuespecimen.

It is a further object of the present invention to provide a cassettefor retaining a tissue specimen which enables Mohs surgery by opticalsectioning the specimen with a confocal microscopic or other opticalimaging microscope.

It is a still another object of the present invention to provide acassette for a tissue specimen which can be used to archive and identifythe tissue specimen more easily than the prior art tissue specimens fromMohs surgery which are physically sectioned and prepared on multipleslides.

It is yet another object of the present invention to provide a methodfor using a cassette to prepare a tissue specimen for examination by aconfocal microscope or other optical imaging microscope.

A still further object of the present invention is to provide a cassettefor retaining a tissue specimen which allows the entire sample to beobservable in the cassette through the top and bottom of the cassette.

Briefly described, the present invention embodies a cassette having abase member with a rigid optically transparent window upon which atissue specimen is situated, a pliable plastic membrane which islocatable over a substantial portion of the base member including thewindow, and an upper member, having an aperture therethrough, locatableover the base member to provide an enclosed cavity between the membraneand the window sealing the tissue specimen therein. With the tissuespecimen in the cavity, the edges of the tissue specimen may bepositioned through the aperture of the upper member and the membranesuch that they lie planar against the window. The edges may be retainedin that position by multiple bonds formed between the membrane andwindow at points or locations around the tissue specimen. The specimenis observable through the aperture of the upper member and imagible byan optical imaging microscope through the window of the base member.

The base member may have at least one injection port through whichfluids, via a syringe needle, may be inserted and removed from thetissue specimen retained in the cavity of the cassette. Such fluids canfacilitate imaging of the specimen by the optical imaging microscope, orcan be used to place the specimen in a preservative for archivingpurposes. A label with indicia identifying the tissue specimen may beapplied to the cassette.

To form the cavity containing the tissue specimen, the lower surface ofthe upper member may have an adhesive, such as double-sided tape, whichproduces a seal between the upper and base members when the upper memberis located over the base member. In another embodiment, the base membermay have walls extending about its periphery into which the upper membermay be received. The upper member has an annular ridge extending alongits outer edge which is received in an annular grove along the inside ofthe wall of the base member, thereby forming a seal between the upperand base members.

When the cassette is closed, i.e., the tissue specimen is sealed in thecavity between the membrane and window, the membrane is held tightly bythe upper member against the base member over the tissue specimen. Thepressure of the membrane may compress the tissue specimen toward thewindow of the base member. The top of the tissue specimen can be cut orscored prior to closure of the cassette to facilitate movement of theedges of the tissue specimen toward the window when the specimen iscompressed by the membrane. A user, such as a physician or trainedoperator, with a first probe may manipulate under the tension of themembrane each of the edges of the specimen planar against the window,and then with a second probe retain the edge in that planar position bybonding the membrane and window together at one or more points near thespecimen's edge. To produce a bond at each point, the second probepushes the membrane adjacent the window and then conducts heat to jointhe window and membrane together. Thus, the edges of the tissue specimenare oriented planar against an optically transmissive surface providedby the window of the cassette. Fluids can be inserted and removed fromthe tissue specimen through the injection port, since the multiple bondsdo not seal the specimen within the cavity between the membrane and thewindow. Other bond actuating means may also be used such as sonicwelding, or by the use of a contact, or UV cure, adhesive. Such anadhesive may be applied to lower surface of the membrane or uppersurface of window facing the membrane, or both, prior to placement ofthe tissue specimen in the cassette, such that contact of the membraneand window by the second probe forms an adhesive bond.

The cassette having a properly oriented tissue specimen may be part of aconfocal imaging system for producing microscopic images of sections ofthe tissue contained in the cassette. The system includes a confocalimaging head, a stage supporting the cassette which presents the windowof the cassette to an objective lens of the confocal imaging head, and acamera which can capture images of the specimen through the aperture ofthe cassette's upper member. A control system is coupled to a display,the confocal imaging head and the camera to visualize on the displayboth images of microscope sections of the tissue specimen produced bythe confocal imaging head and macroscopic images of the tissue capturedby the camera. During examination of the tissue specimen in thecassette, the images from the camera can be used to identify thelocation of the microscopic images of sections with respect to thespecimen.

The present invention further embodies a method using a cassette forpreparing a tissue specimen for examination by a confocal microscope orother optical imaging microscope. The method includes the steps oflocating a tissue specimen on a rigid optically transparent window of abase member, placing a pliable optically transparent membrane over atleast a substantial portion of the base member including the window,sealingly engaging the membrane to the base member to produce a cavitybetween the membrane and the window, positioning edges of the tissuespecimen against the window, and fixing the location of the edgespositioned against the window by connecting the membrane to the windowat multiple points.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing objects, features and advantages of the invention willbecome more apparent from a reading of the following description inconnection with the accompanying drawings, in which:

FIGS. 1 and 1A are perspective views of an open cassette in accordancewith the present invention in which FIG. 1 shows the membrane attachedto the upper member of the cassette, and FIG. 1A shows the membraneseparate from the upper member of the cassette;

FIG. 1B is cross-sectional view along line 1B-1B of FIG. 1 showing theinjection port of the cassette of FIG. 1 in more detail;

FIG. 1C is a partial cross-sectional view from the edge of the cassettealong line 3-3 of FIG. 1 showing an alternative side injection port;

FIG. 2 is a perspective view of the cassette of FIG. 1 or FIG. 1A whenclosed;

FIG. 3 is cross-sectional view along line 3-3 of FIG. 1 showing thewindow of the base member of the cassette without a tissue specimen;

FIG. 4 is cross-sectional view along line 3-3 of FIG. 1 showing anotherembodiment of the window of the base member of the cassette without atissue specimen;

FIG. 5 is cross-sectional view along line 3-3 of FIG. 1 showing anotherembodiment of the base member without the tissue specimen in which thebase member is composed of an optically transparent material to providethe window of the cassette when the cassette is closed;

FIG. 6 is a cross-sectional view similar to FIG. 3 showing the movementof the upper member and the membrane over the base member of thecassette of FIG. 1A from an open cassette to a closed cassette;

FIG. 7 is a cross-sectional view along line 7-7 of FIG. 2 showing thetissue specimen sealed in a cavity between the membrane and the windowof the cassette;

FIG. 8 is a cross-section view similar to FIG. 7 showing an example of afirst probe positioning an edge of the tissue specimen in a planarorientation against the window of the cassette;

FIG. 9 is a cross-section view similar to FIG. 8 showing an example of asecond probe for bonding the membrane and window together at a locationto retain the planar orientation of the edge of the tissue specimen;

FIG. 10 is a cross-section view similar to FIG. 9 showing an example ofthe tissue specimen with its edges positioned planar against the windowand the membrane bonded to the window at multiple locations around thetissue specimen;

FIG. 11 is a bottom view of the cassette of FIG. 10 showing theinjection port of the cassette and an example of a tissue specimen inthe cassette with the edges of the specimen positioned planar againstthe window by multiple bonds at locations around the tissue specimen;

FIG. 12 is a cross-sectional view along line 12-12 of FIG. 11 showing anexample of a syringe needle inserting fluid into the cavity of thecassette through the injection port;

FIG. 12A is a cross-sectional view along line 12-12 of FIG. 11 showingan example of side injection ports to the cavity of the cassette;

FIG. 13 is a perspective view of the cassette in accordance with anotherembodiment for sealing the upper member and base member of the cassettetogether;

FIG. 13A is cross-sectional view along line 13A-13A of the cassette ofFIG. 13;

FIG. 13B is cross-sectional view along line 13A-13A of the cassette ofFIG. 13 showing two injection ports;

FIG. 13C is cross-sectional view along line 13A-13A of the cassette ofFIG. 13 showing an example of a side injection port of FIG. 1C;

FIG. 14 is a block diagram of a confocal microscope system with acassette in accordance with the present invention;

FIG. 15 is an illustration of a screen on the display of the system ofFIG. 14 during confocal imaging of a tissue specimen in the cassette;and

FIG. 16 is an enlarged view illustrating the corrugations formed betweena tissue specimen and the window of the cassette.

DETAILED DESCRIPTION OF INVENTION

Referring to FIGS. 1-3, a cassette 10 of the present invention is shownhaving a base member 12 and an upper member 14. Base member 12 has anaperture 18 and a rigid optically transparent window 16 situated inaperture 18. The upper member 14 has an aperture 20 preferablysubstantially greater in size than the aperture 18 of base member 12.Base and upper members 12 and 14 are hinged at a hinge 24 which allowscassette 10 to have an open state, as shown in FIG. 1, and a closedstate, as shown in FIG. 2, where upper member 14 covers base member 12.Although preferably base and upper members 12 and 14 are attached by ahinge 24, they may, in the alternative, be separate and unattached toeach other when cassette 10 is in an open state.

As best shown in FIG. 3, at the bottom surface 12 a of base member 12,the base member has an annular shelf 22 in communication with aperture18. Window 16 is inset on shelf 22, such that bottom surface 12 a of thebase member is planar with the lower surface 16 a of window 16. Window16 may be held by an adhesive, such as glue, upon shelf 22, or may beinsert molded with base member 12. In the alternative, window 16 may beattached to bottom surface 12 a underneath aperture 18 without shelf 22,such as by an adhesive or glue along the part of bottom surface 12 awhich interfaces with window 16, as shown in FIG. 4. The window 16 maybe composed of a cut out sheet of thin glass or amorphous polyolefin,such as Zeonex plastic, or other single layer optically homogeneousmaterial through which optical imaging can be performed. Preferably,window 16 is of 0.006 inch thick Zeonex plastic film which is rigidlyattached to the base member 12 in either the configuration of FIG. 3 or4. The attachment of window 16 in cassette 10 should not be limited tothat described herein. Any means for attaching an optical transparentwindow to form part of base member 12 may be used. The thickness of thewindow 16 depends on the working distance of the optical image systemwhich will image the tissue specimen through the window. In a furtheralternative shown in FIG. 5, the base member 12 may itself be composedof an optically transmissive material, such as 1 millimeter thick Zeonexplastic providing a single layer of material, without aperture 18.Window 16 in FIG. 5 represents the part of the base member 12 belowaperture 20 when the cassette 10 is in a closed state.

A pliable plastic membrane or film 26 (FIGS. 1 and 1A) is attached tobottom surface 14 a of upper member 12 across aperture 20. Membrane 26does not extend to the outer edge 14 b of upper member 14, therebyleaving a region 14 c near the outer edge of the upper member. Anadhesive, such as double-sided tape, may fix membrane 26 to upper member12 along bottom surface 14 a outside region 14 c. Optionally, membrane26 may be separate from upper member 12, as shown in FIG. 1A, in whichthe membrane is held along one side by hinge 24. The membrane 26 may be,for example, a thin layer of plastic, such as plastic wrap typicallyused for food preservation, and should be sufficiently transparent toprovide viewing therethrough by a user or camera.

Base member 12 further includes an injection port 28, as best shown inFIG. 1B. The injection port 28 is defined by an opening 28 a whichextends from the lower surface 12 a of the base member 12 partiallythrough the base member, a channel 28 b which extends from the opening28 a to the upper surface 12 b of the base member 12, and a self-sealingmember 28 c which is received in opening 28 a from the bottom surface 12a of the base member. Between self-sealing member 28 c and channel 28 bis a passageway 28 d in communication with channel 28 b. Self-sealingmember 28 c may be composed of rubber, and may be similar to theself-sealing members used with medical vials for enabling a syringeneedle to be inserted without loss of vial containment after removal ofthe syringe needle. An alternative side injection port 33 from the sideof base member 12 is shown in FIG. 1C. Injection port 33 has an opening33 a partially through the base member, a channel 33 b which extend fromopening 33 a to the wall of aperture 18, a self-sealing member 33 cwhich is received in opening 33 a, and a passageway 33 d between theself-sealing member 33 c and channel 33 b. As will be described later, asyringe needle may be inserted into the injection port 28 or 33 throughself-sealing member 28 c or 33 c to passageway 28 d or 33 d to insert orremove fluid from a cavity in the cassette 10 containing the tissuespecimen, via channel 28 b or 33 b, when the cassette 10 is in a closedstate. Although only one injection port is illustrated in FIGS. 1, 1Band 1C, multiple ones of the injection ports may be provided in cassette10.

The base and upper members 12 and 14 may be composed of rigid material,such as plastic. Hinge 24 may also be made of plastic, or can beprovided by a strip of adhesive material, such as tape, along the oneside of members 12 and 14. Member 12 and 14, and hinge 24 may be asingle molded piece, or separately molded. The dimensions of thecassette 10 are such that window 16 has a diameter larger than the widthof the tissue specimen to be located on the window. For example, thecassette may have an aperture 18 having a 2 cm diameter and a 3 mm depthto window 16, and when closed the cassette may be 2.5 cm in length by2.5 cm in width with a height of 7 mm. The cassette 10 may be largerthan these dimensions to accommodate larger tissue specimens. Apertures18 and 20 are circular, but may be rectangular, or other shape.

In an open state, i.e., an open cassette 10, a surgically exposed tissuespecimen 13 is situated on window 16, as shown for example in FIGS. 1and 1A. Typically, the tissue specimen is elliptical in shape withcurved, sloping sides. With the tissue specimen 13 on window 16, uppermember 14 is located over the base member 12 to cover the base member,such that membrane 26 lies between the base member and upper member overaperture 18 of the base member. The cassette 10 in this closed state,i.e., a closed cassette, is shown for example in FIG. 2. In the exampleof FIG. 1A, FIG. 6 shows upper member 14 and membrane 26 rotating alongan arc defined by hinge 24 to provide a closed cassette, as indicated byarrows 30.

In a closed state, a hermetic seal is formed between the upper member 14and base member 12 to define an enclosed cavity or compartment 27between the membrane 26 and window 16 containing tissue specimen 13(FIGS. 2 and 7). The seal may be provided by an adhesive, such asdouble-sided tape, along the region 14 c of upper member 14 (FIGS. 1 and1A). The seal may also be provided by region 14 c of upper member 14having a continuous raised ridge which may be received by correspondingcontinuous groove on the top surface 12 b of the base member 12, suchthat the raised ridge of the upper member snap fits in the groove of thebase member. Other sealing means may alternatively be used, such as agasket between the upper and base members, UV cure adhesive, sonicwelding, or thermal welding.

In the closed cassette, the tissue specimen 13 is observable throughboth window 16, and membrane 26 through aperture 20 of upper member 14.Although aperture 20 is preferably larger than aperture 18, it may bethe same size or smaller than aperture 18 so long as when the cassetteis closed both the tissue specimen 13 a gap or space 29 (FIG. 2) isviewable around the edges 13 a of the tissue specimen through aperture20. Further, with the tissue specimen 13 sealed in cavity 27, theinjection port 28 (and/or injection port 33) enables fluids to beinserted or removed from the tissue specimen retained in the cavity 27.A label 31 with indicia identifying the tissue specimen 13 may beapplied to the top surface 32 of the cassette 10, or along the bottomsurface 12 a of cassette 10, or both, such as shown in FIGS. 2 and 11.The indicia may represent a bar code or alphanumeric numeralsidentifying the tissue specimen, such as the patient name, date,physician, or other references to the surgical procedure.

Referring to FIGS. 7-11, the orientation of the tissue specimen 13 onwindow 16 in a closed cassette will now be described. If prior toclosure of cassette 10, the tissue specimen 13 on upper surface 16 b ofwindow 16 in aperture 18 extends beyond the top surface 12 b of basemember 12, membrane 26 compresses the tissue specimen, as shown in FIG.7. The top of the tissue specimen 13 may be scored prior to the closureof the cassette 10 to facilitate movement of the edges 13 a of thetissue specimen 13 toward window 16. One or more scoring incisions alongthe top surface of the tissue may be needed when the specimen's edgesare at a steep angle with respect to the surface of window 16. Suchincisions should not extend through the specimen, and may be in across-pattern or other pattern which relaxes tension of the tissue suchthat the tissue edges may be moved to flatten them against window 16.

As shown in FIG. 8, a user, such as a physician or trained operator,using a probe 34 manipulates one of the edges 13 a of the tissuedownwards against the window 16 under the tension of membrane 26, suchthat the edge is planar with upper surface 16 b of window 16. With theedge 13 a held in that position, the user manipulates another probe 36against the membrane 26 in gap 29 until reaching window 16 at a locationnear the first probe which will hold the edge 13 a in the desireposition when probe 34 is removed, as shown in FIG. 9. The probe 36 isthen actuated by the user to conduct heat though thermal bond actuatingmeans 38 to produce a bond (weld or joint) between the membrane 26 andthe window 16 at a point or location 40 (FIG. 10), thereby retaining theedge 13 a of the specimen in the desired planar orientation againstwindow 16. Thermal bond actuating means 38 may be similar to a solderingiron operating at a low temperature to weld the material of the plasticmembrane and window together without affecting the integrity of thecavity. Other bond actuating means may also be used, such as sonicwelding. If needed, more than one bond at different locations 40 in gap29 may be used to retain edge 14 a. This is repeated around the tissuespecimen 13 until all of the edges 13 a are held in a planar orientationagainst the window 16 by multiple bonds 40 between membrane 26 andwindow 16. FIGS. 10 and 11 show multiple bonds 40 retaining the tissuespecimen 13 in the desired planar orientation. Probes 34 and 36 havesufficiently blunt ends to avoid puncturing membrane 26 during thisprocedure. In this manner, tissue specimen 13 is oriented in a closedcassette 10 such that the edges 13 a of the tissue specimen arepositioned planar against the window 16 and are retained in thatposition by bonds formed between the membrane 26 and window 16 atmultiple points or locations 40 around the tissue specimen.

In the alternative to thermally or sonically formed bonds, a contactadhesive may be applied to the upper surface 16 a of window 16 or thesurface of membrane 26 facing window 16, or both, prior to placement ofthe tissue specimen in the cassette, such that probe 36 by contactingmembrane 26 to window 16 adhesively bonds them to each other at a point40. Further, the contact adhesive may be a UV light cure adhesive, suchthat after the bonds are formed they may be exposed to UV light toharden them.

Referring to FIG. 12, a syringe (not shown) with a needle 42 may beinserted into injection port 28 through self-sealing member 28 c toforce fluid into cavity 27 through passage 28 d and channel 28 b. Sincemultiple bonds do not seal tissue specimen 13 within cavity 27, thefluid freely flows to the tissue specimen 13, thereby immersing thetissue specimen in fluid, as shown by arrows 44. Thus, such bonds joinmembrane 26 and the window 16 in a non-sealing relationship with respectto the specimen 13 to allow fluid flow. The fluid may represent anoptical index matching fluid to facilitate imaging of the specimen by anoptical imaging microscope. Similarly, the fluid may be withdrawn by theuse of a syringe with needle 42 to suction the fluid from cavity 27. Theself-sealing member 28 c allows the insertion of the needle withoutcompromising the integrity of the cavity 27. Thus, the tissue specimenis retained in the cassette in the desired orientation, while beingpermeable to fluids inserted through the injection port 28. The fluidinserted flows between the tissue specimen 13 and window 16 due to theelasticity of the membrane 26 and the pressure of the fluid.

For the side injection port 33 of FIG. 1B, FIG. 12A shows the needle 42inserted into injection port 33 through self-sealing member 33 c toinsert fluid to, or withdraw fluid from, cavity 27 through passage 33 dand channel 33 b. FIG. 12A also shows another side injection port 33 aidentical to injection port 33, but oriented opposite injection port 33in base member 12, such that the cassette has two injection ports 33 and33 a. This may facilitate the flow of fluid through cavity 27 bysimultaneously inserting a fluid through port 33 and removing the fluidthrough port 33 a, or vise versa. The second injection port 33 a mayalso serve as a backup port if the first injection port 33 fails tofunction.

Referring to FIGS. 13 and 13A, another embodiment is shown for sealingthe upper and base members of cassette 10 together. In this embodiment,the base and upper members of cassette 10 are denoted as 46 and 48,respectively, and the base member of FIG. 5 is shown. Base member 46 hasan annular wall 46 a which extends about its periphery into which theupper member 48 can be received. The upper member 48 has an aperture 49and an annular ridge or tongue 48 a which extends along its outer edge.Upper member 48 is insertable into the base member 46 such that ridge 48a fits into an annular groove 46 b of base member 46 along the inside ofwall 46 a, thereby sealing the upper member 48 to the lower member 46 todefine an enclosed cavity 56 between the membrane 53 and the base member46. The compression of the edges 13 a of the tissue specimen orientssuch edges planar against a window 54, which is defined by the part ofthe optically transmissive base member 46 below aperture 49 of uppermember 48. Upper member 48 is attached to base member 46 by a loopmember 52, which may be composed of plastic. Aperture 49 may be sizedsufficiently larger that the tissue specimen 13 to enable multiple bondsto be made between membrane 53 and window 54 to hold the edgespositioned planar to the window, such as described earlier. Membrane 53is similar to membrane 26, and injection port 50 is similar to injectionport 28 with a self-sealing member 59 and a channel 58 through wall 46 ato enable fluids to be insertable and removable from the tissue specimen13 in cavity 56. Upper member 48 may have circular or disc-like shapewith a circular, curved lower surface 48 b.

Referring to FIG. 13B, in the embodiment shown in FIG. 13, a secondinjection port 50 a may be provided from the top surface of base member46 in addition to, or instead of, injection port 50 Like injection port50, injection port 50 a 50 has a self-sealing member 59 a and a channel58 a through wall 46 a to enable fluids, via a syringe needle, to beinsertable and removable from the tissue specimen 13 in cavity 56. Thetwo ports 50 and 50 a have channels 58 and 58 a, respectively, which maybe located along opposite sides of base member 46. By providing acassette 10 with two injection ports, fluid can be simultaneouslyinserted through one port and removed through the other port to enable afluid flow through cavity 56. Although only one or two ports are shown,additional number of ports may be similarly provided at differentlocation in the base member. A variation of the injection port 50 and 50a is shown in FIG. 13C in which a side injection port 50 b is providedthrough the side of base member 46. Like injection ports 50 and 50 a,injection port 50 b has a self-sealing member 59 b and a channel 58 bthrough wall 46 a to enable fluids, via a syringe needle, to beinsertable and removable from the tissue specimen 13 in cavity 56.Multiple ones of side injection port 50 b, such as two, may be providedat opposite sides of the base member 46.

Referring to FIG. 14, a confocal imaging system 60 having the cassette10 is shown. Confocal imaging system 60 includes a computer controlsystem 62 having a confocal imaging head 64 with a liquid immersionobjective lens 64 a, and a display 66 and user interface 68 coupled tocontrol system 62. The confocal imaging head 64 and control system 62provides confocal microscopic images of optically sectioned tissue onthe display 66 as described, for example, in U.S. Pat. No. 5,788,639, orin published International Patent Application WO 96/21938, which areherein incorporated by reference. The confocal imaging head 64, controlsystem 62, display 66, and user interface 68 may represent a confocalmicroscope, such as the “VivaScope” manufactured by Lucid, Inc. ofHenrietta, N.Y.. A z-actuator 70 can move the confocal imaging head 64(or alternately, only objective lens 64 a) in a z direction, via signalsfrom control system 62, to control the depth of imaging in tissue, i.e.,the distance of objective lens 64 a to window 16 of cassette 10.

The confocal imaging system 60 further includes an x-y stage 72 whichsupports cassette 10 and presents the window 16 of the cassette to aliquid immersion objective lens 64 a of the confocal imaging head 64.Stage 72 has actuators (not shown) which can move the cassette 10 in x,yorthogonal directions in response to signals from control system 62.User interface 68 may represent a keyboard, mouse, joystick, orcombinations thereof, which enable a user, via control system 62, tocontrol the z-actuator 70 and x-y stage 72 such that different parts ofthe tissue specimen may be imaged by the confocal imaging head 64. Priorto locating the cassette in stage 72, a fluid which is matched to theoptical index of the immersion objective lens 64 a is inserted into thecavity of the cassette 10 via the injection port 28 or 33. Optionally,an optical coupling fluid 71 may be placed between lens 64 a and window16 of cassette 10 to optically couple the lens 64 a to window 16. Thefluid 71 may have the same optical index as the fluid inserted in thecavity of cassette 10. The cassette 10 may that than shown in FIG. 11 or13.

An optional camera 74 in the confocal imaging system 60 is provided tocapture images of the tissue specimen through a lens 74 a directed toaperture 20 in upper member 14 of the cassette 10. Camera 74 may be adigital camera which captures still images, or may be a video camera.The control system may also send signals to the camera 74 to control itsoperation, such as enabling the camera to capture an image or focusingthe camera. Signals representing captured images from camera 74 arereceived by the control system 62.

On display 66 the control system 62 visualizes images of microscopicsections of the tissue specimen from confocal imaging head 64 andmacroscopic images from camera 74. FIG. 15 shown an example of a screen(denoted as 75) of display 66 during confocal imaging, where image 76represents a macroscopic image of a tissue specimen in cassette 10 fromcamera 74, and image 78 represents an optical section of the tissuespecimen imaged through confocal imaging head 64. Image 76 may bemaintained on the screen 75 during optical sectioning to guide the useras to the location of microscopic section 78 with respect to the tissuespecimen. For example, a box 77 may indicate the relative location ofthe microscopic image 78 with respect to the macroscopic image 76. Thex-y stage 72 is positioned with respect to camera lens 74 a such that amarker, such as a cross-hair, may be overlaid by control system 62 onmacroscopic image 76 showing the relative location of the confocalimaging head 64 with respect to the tissue specimen. Since the edges ofthe tissue specimen are planar against window 16 of cassette 10, opticalsections imaged on display 66 from along the edges of the tissuespecimen can provide information determining whether a tumor is fullycontained in the tissue specimen for Mohs surgery. If desired, images ofoptical sections from different parts of the tissue specimen may bescanned automatically by the control system 62 by controlling themovement of x,y stage 72. The scanned images of optical sections areelectronically combined in memory of the control system 62 to provide anoptical section having a larger field of view to the user. If imaging byanother confocal imaging head is desired, the fluid can be replaced witha different optical index matching fluid, via injection port 28 of thecassette.

Although a confocal imaging head 64 is described herein, other opticalimaging techniques may also be used in head 64, such as opticalcoherence tomography, such as described in Schmitt et al., “Opticalcharacterization of disease tissues using low-coherence interferometry,”Proc. of SPIE, Volume 1889 (1993), or a two-photon laser microscope, asdescribed in U.S. Pat. No. 5,034,613.

After imaging of the tissue specimen is complete, the tissue specimencan be removed from stage 72 and archived in cassette 10. To preservethe tissue specimen, a preservative fluid, such as formalin, may beinserted in the cavity of the cassette, via injection port 28, after anyfluid in the cavity is removed. The entire cassette with the tissuespecimen from Mohs surgery can thus be stored intact with indicia 31 onthe cassette referencing the procedure. Optical sectioning of tissuespecimens can facilitate their archiving since intact specimens mayrequire less storage space than traditional slides, and can easily belabeled with indicia 31 on the cassette.

The particular immersion fluid inserted through an injection port intothe cavity of the cassette 10 containing the tissue specimen 13 may beselected as follows to enhance imaging. Surface corrugations at theinterface between the tissue specimen 13 and the window 16 are filledwith the immersion fluid, which produces optical corrugations in thewavefront of the beam focused into the specimen. These corrugationsreduce the fidelity of the images. The effect of the corrugations can bereduced by matching the refractive index of the immersion liquid withthe tissue. As shown in FIG. 16, the corrugations due to the surfacetexture of the specimen 13 creates corrugations having a depth (h) (fromthe apex of the corrugation peaks to the bottom of the valleys of thecorrugations) which may be approximately 200 microns in length. Theindex of refraction of the tissue is n_(T), while the index ofrefraction of the immersion fluid, which fills the corrugationsproviding the surface texture of the specimen 13, have an index n_(I).The beam is focused at a focus fin the section to be imaged. Thewavefront which may be spherical, can be distorted due to an opticalpath difference φ imprinted on the wavefront which converges to thefocus F. This path difference is a function of the product of thecorrugation height h and the difference between n_(T) and n_(I). The useof the index matching fluid reduces the optical path difference so thatthe imprint is minimized. The optical path difference φ is shownenlarged at 80 in FIG. 16. This optical path difference may also beviewed as the wavefront which is propagating to the focus F. Thiswavefront may be spherical and part of a sphere as shown at 82 prior topassing through corrugations at the surface of the specimen 13. Theoptical path distortion after transmission through the tissue surface isapproximated by the relation φ=h (n_(T)−n_(I)), where h is themechanical depth of surface texture. In order to correct for thedistortion of the beam wavefront (which may be a spherical wavefront) byvirtue of the variation in index of refraction presented bycorrugations, it is desirable that the difference in index of theimmersion liquid 84 and the average index of refraction of the tissuemultiplied by the corrugation height h that is the optical path distancebetween the hills and valleys of the corrugation), not exceed a quarterwavelength of the laser beam which is used for imaging by the imagingsystem 62. Thus, the immersion medium 84 is selected for the tissue typewhich is placed in the cassette and substantially corrects for opticaldistortion due to the surface texture of the tissue specimen.

From the foregoing description, it will be apparent that there has beenprovided a cassette for facilitating optical sectioning of a retainedtissue specimen. Variations and modifications in the herein describedcassette, method, and system in accordance with the invention willundoubtedly suggest themselves to those skilled in the art. Accordingly,the foregoing description should be taken as illustrative and not in alimiting sense.

What is claimed is:
 1. A retaining device for a tissue specimen having ashape with curved sides to enable optical sectional imaging of tissueedges along said sides, said retaining device comprising: a rigid windowof a single layer of transparent material imagible there through, saidwindow having a tissue specimen support surface directly contacting thetissue specimen when thereupon; and a member locatable over at leastsaid window of a different material than said window in which saidmaterial of said member applies pressure onto one or more curved sidesof a tissue specimen when present on said tissue specimen supportsurface so that tissue edges along the one or more curved sides of thetissue specimen lie against said surface.
 2. The device according claim1 wherein said window and said member are of a substantially differentthickness.
 3. The device according claim 1 wherein said memberrepresents a first member, and said retaining device further comprisinga second member and said window is one of an optically transmissive partof said second member or optically transmissive material adjoining saidsecond member.
 4. The device according to claim 1 wherein said tissuespecimen is imagible by an imaging system operating in accordance withone of two-photon microscopy, optical coherence tomography, or confocalmicroscopy.
 5. A device for a tissue specimen for examination by anoptical microscope for optically imaging sections comprising: a firstmember; a window having a single layer of rigid optical transparentmaterial adjoining said first member and a tissue specimen supportsurface; and a second member locatable at least over said window, inwhich the tissue specimen is locatable between said second member andsaid window, and said second member is substantially pliable in responseto pressure to enable said second member to contact said tissue specimensupport surface at one or more locations to orient at least one edge ofthe tissue specimen which is substantially non-planar with respect tosaid window and hence not imagible by an optical sectioning microscopevia said window to a substantially planar position against said surfaceto enable imaging of at least said edge by the microscope via saidwindow exclusive of said first member and said second member beingimaged through, and when said window is imaged through the same saidfirst member adjoins said window as when said second member was locatedover said surface of said window.
 6. The device according to claim 5wherein said microscope operates in accordance with one of two-photonmicroscopy, optical coherence tomography, or confocal microscopy.
 7. Thedevice according to claim 5 further comprising one or more bonds betweensaid second member and said window capable of retaining the tissuespecimen in a certain orientation against said window.
 8. The deviceaccording to claim 5 wherein said window is one of a transparent thirdmember located in an aperture of said first member, or represents a partof said first member being optically transmissive.
 9. A retaining devicefor a tissue specimen comprising: a member, and a rigid window having atissue specimen support surface, said window being of a single layerrepresenting one of an optically transmissive part of said member oroptically transmissive material adjoining said member; and a layer ofmaterial locatable over at least said surface of said window, in whichthe tissue specimen is locatable between said layer of material and saidsurface, and said layer of material is substantially pliable in responseto pressure to shape the tissue specimen upon said surface.
 10. Thedevice according to claim 9 wherein said member responsive to pressureat one or more selected locations enables said layer of material tocontact said surface of said window at said one or more selectedlocations so that the tissue specimen has a selected shaped orientation,and at each of said one or more selected location said layer of materialin response to an external applied means forms a bond between said layerof material and said window to retain said specimen in said selectedshaped orientation.
 11. The device according to claim 10 wherein saidexternal applied means is provided by at least a thermal or sonicwelding element to form said bond at each of said one or more selectedlocations.
 12. The device according to claim 10 further comprisingadhesive material between said member and said window, wherein saidmember responsive to pressure enables said layer of material to contactsaid surface of said window at one or more selected locations to enablethe tissue specimen to have a selected shaped orientation upon saidsurface, and said adhesive material bonds said layer of material to saidsurface at each of said one or more location so as to enable saidspecimen to maintain said selected shaped orientation.
 13. The deviceaccording to claim 12 wherein said adhesive material is curable byultraviolet (UV) light, and at each of said one or more selectedlocation said layer of material provides material which in response toapplied UV light forms said bond between said layer of material and saidsurface of said window to retain said specimen in said selected shapedorientation.
 14. The device according to claim 9 wherein said layer ofmaterial is a thin layer of plastic material.
 15. The device accordingto claim 9 wherein said window enables the tissue specimen to beimagible via said window to a depth in the tissue specimen to enableimaging of one or more optically form a microscopic sectional images atsaid depth in the tissue specimen.
 16. The device according to claim 9wherein said layer of material contacts said member.
 17. The deviceaccording to claim 9 wherein said window is substantially thicker thansaid layer of material.
 18. The device according to claim 9 wherein saidmember has an aperture and said window when adjoining said member islocated in or over said aperture of said member and rigidly attached tosaid member.
 19. A method for using a cassette for preparing a tissuespecimen for examination, comprising the steps of: locating a tissuespecimen on a rigid window composed of a single layer of opticallytransparent material; and placing a membrane over at least said surfaceof said window to retain the tissue specimen against said surface, inwhich said membrane, and said membrane is sufficiently pliable inresponse to pressure to be operated upon to reach said surface at one ormore locations to shape the tissue specimen upon said surface.
 20. Themethod according to claim 19 further comprising the steps of: applyingselected pressure to one or more locations upon said membrane to shapethe tissue specimen upon said surface of said window; and joining saidmembrane to said surface at each of said one or more locations when saidapplied pressure enables said membrane to contact said window so as toenable said tissue specimen to retain the shape responsive to saidapplied pressure.
 21. The device according to claim 1 further comprisingone or more bonds which are formed between said member and said windowto join said member to said window in a non-sealing relationship withrespect to the tissue specimen.